This application is a national stage filing under 35 U.S.C. xc2xa7371 of international application No. PCT/EP99/01113, filed on Feb. 20, 1999.
Process for the enzymatic separation of enantiomers of 3(R)- and 3(S)-hydroxy-1-methyl-4-(2,4,6-trimethoxyphenyl )-1,2,3,6-tetrahydropyridine or of the carboxylic acid esters.
The invention relates to a process for the preparation of optically pure compounds of the formula (I) by stereodifferentiating reaction of the mixtures of enantiomers with the aid of an enzyme.
3(S)- and 3(R)-hydroxy-1-methyl4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetrahydropyridine (compounds of the formula (I) where Rxe2x95x90H) or their ester derivatives (compounds of the formula (I) where Rxe2x95x90COR1) are central units or precursors of the synthesis of flavopiridol (HMR 1275 or L 86 8275) described in the patent application No. HMR 98/L 001 (xe2x80x9cProcess for the preparation of (xe2x88x92)cis-3-hydroxy-1-methyl-4(R)xe2x88x92(2,4,6-trimethoxy-phenyl)piperidine)xe2x80x9d), of the first potent inhibitor of cyclin-dependent protein kinase (see, for example, Sedlacek, Hans Harald; Czech, Joerg; Naik, Ramachandra; Kaur, Gurmeet; Worland, Peter; Losiewicz, Michael; Parker, Bernard; Carlson, Bradley; Smith, Adaline; et al. Flavopiridol (L 86 8275; NSC 649890), a new kinase inhibitor for tumor therapy, Int. J. Oncol. (1996), 9(6), 1143-1168 or Czech, Joerg; Hoffmann, Dieter; Naik, Ramachandra; Sedlacek, Hans-Harald; Antitumoral activity of flavone L 86 8275. Int. J. Oncol. (1995), 6(1), 31-36).
A resolution of racemates or separation of enantiomers of the compounds of the formula (I) is not known.
It has now been found that compounds of the formula (I) can be obtained in optically pure form from the mixtures of enantiomers by enzymatic ester cleavage (hydrolysis or alcoholysis).
The present invention thus relates to a process for the kinetic resolution of racemates of compounds of the formula (I), 
which comprises subjecting enantiomer mixtures or racemic mixtures of compound s of the formula (I), in which
R is COR1 where R1=(C1-C16)-alkyl, (C2-C16)-alkenyl or (C3-C16)-alkynyl, CnH2n-cycloalkyl where n=1-16, which can be branched or unbranched and which can be substituted by 1-3 substituents from the group F, Cl, Br, I, CF3, CN, NO2, hydroxyl, methoxy, ethoxy and COOR2, where R2=(C1-C4)-alkyl and (C2-C4)-alkenyl, which can be branched or unbranched and which can be substituted by 1-3 substituents from the group consisting of F, Cl, Br, CF3,
in homogeneous or heterogeneous, aqueous, aqueous/organic or organic media in the presence of an enzyme, e.g. of a lipase or esterase, e.g. from mammalian livers or pancreases or microbial origin, such as, for example, from Candida, Pseudomonas and Aspergillus, or of a protease, e.g. from Bacillus, to a stereoselective hydrolysis or alcoholysis at a temperature of 10-80xc2x0 C., if appropriate in the presence of cosolvents and of a buffer, the reaction mixture preferably containing 2-50% by weight of ester
and, after the reaction has taken place, separating the unreacted ester (compound of the formula (I) where Rxe2x95x90COR1) and the alcohol formed (compound of the formula (I) where Rxe2x95x90H)xe2x80x94and thus the two enantiomers.
The process according to the invention is economical, simple and rapid. The reaction does not require any equimolar amounts of optically pure auxiliaries, any expensive reagents, any disproportionately large amounts of solvent and any cost-intensive working steps. After the completion of the reaction, the separation of the products or of the enantiomers can be carried out by simple measures, e.g. by extraction.
Preferably, in the compounds of the formula (I)
R is COR1 where R1=(C1-C12)-alkyl, (C2-C12)-alkenyl or (C3-C12)-alkynyl, CnH2n-cycloalkyl where n=1-12, which can be branched or unbranched and which can be substituted by 1-3 substituents from the group consisting of F, Cl, Br, CF3, CN, NO2, hydroxyl, methoxy, ethoxy and COOR2, where R2=methyl, ethyl and vinyl, which can be substituted by 1-3 substituents from the group consisting of F, Cl, CF3.
Particularly preferably, in the compounds of the formula (I)
R is COR1 where R1=(C1-C10)-alkyl, (C2-C10)-alkenyl or (C3-C10)-alkynyl, CnH2n-cycloalkyl where n=1-10, which can be branched or unbranched and which can be substituted by 1-3 substituents from the group consisting of F, Cl, Br, CF3, CN, NO2, methoxy, and COOR2, where R2=methyl, ethyl and vinyl, which can be substituted by 1-3 substituents from the group consisting of F, Cl, CF3.
Very particularly preferably, in the compounds of the formula (I)
R is COR1 where R1=(C1-C10)-alkyl, (C2-C10)-alkenyl or (C3-C10)-alkynyl,
which can be branched or unbranched and which can be substituted by 1-3 substituents from the group consisting of F, Cl, Br, CF3, and methoxy.
A procedure is preferably used in the process in which an ester of the formula (I), for example R=COR1 where R1=C3H7 or C8H17, is treated with a lipase, esterase or protease in a water- or alcohol-containing solution and stirred. It may be advantageous to buffer the solution mentioned, e.g. with phosphate or TRIS [=tris(hydroxymethyl)-methylamine] buffer. The addition can be, for example, 0.01-1.0 molar. A suitable buffer range is pH 5-9.
It may furthermore be advantageous to add cosolvents. Suitable cosolvents are, for example, dimethoxyethane, acetone, THF, dioxane, hexane, tert-butyl methyl ether and tert-butanol. The proportion of cosolvents in the solution is preferably 10-80%.
The enzymes employed are preferably lipases and esterases, such as, for example, cholesterol esterase (EC 3.1.1.13) from bovine pancreas (Sigma Chemical Co.), porcine liver esterase (PLE, Sigma Chemical Co.), pancreatin (Fluka and Sigma Chemical Co.), pancreas acetone powder from cattle (Sigma Chemical Co.), liver acetone powder from horses (Sigma Chemical Co.) and lipase from porcine pancreas (PPL, Sigma Chemical Co.), lipase OF from Candida rugosa (Meito Sangyo) and lipase AP-6 from Aspergillus niger (Amano Pharmaceuticals).
Each of the enzymes mentioned can be employed in free or in immobilized form (Immobilized Biocatalysts, W. Hartmeier, Springer Verlag Berlin, 1988). The amount of enzyme is freely selected depending on the reaction rate or on the reaction time desired and on the nature of the enzyme (e.g. free or immobilized) and is easy to determine by simple preliminary experiments.
The reaction mixture preferably contains 2-50% by weight of ester, particularly preferably 5-20%. The reaction temperature is 10-80xc2x0 C., preferably 20-60xc2x0 C., particularly preferably 20-40 xc2x0 C.
The preparation of the esters (compounds of the formula (I) where R=COR1) is expediently carried out from the alcohol (compound of the formula I where R=H) according to known methods of esterification (Haslam, Tetrahedron 1980, 36, 2409; Hxc3x6fle, Steglich, Vorbrxc3xcggen, Angew. Chem. 1978, 90, 602) or as described in the patent application HMR 98/L 001 (xe2x80x9cProcess for the preparation of (xe2x88x92)cis-3-hydroxy-1-methyl-4(R)-(2,4,6-trimethoxyphenyl)piperidinexe2x80x9d).
The products resulting from or remaining in the process can be separated in a simple manner, e.g. by extraction or chromatographic methods. The remaining ester is obtained, for example, by partitioning the reaction solution between water and n-heptane and concentrating the organic phase. The resulting alcohol can then be extracted from the aqueous phase with ethyl acetate. The enzyme can be recovered by freeze-drying. The separation (and, if appropriate, later reuse) of the enzyme can be facilitated by immobilization.
By means of suitable conduct of the reaction, it is always possible to obtain at least one enantiomer optically pure. If optically pure ester is desired, the conversion should be over (or equal to) 50%, if optically pure alcohol is desired, the conversion should be smaller (or equal to) 50%. The conversion of the enzymatic hydrolysis or alcoholysis was determined using HPLC (RP 18 LiChrosorb(copyright)) and the determination of the optical purity was carried out by HPLC (Chiralpak AD). The esters resulting from or remaining in the racemate resolution process can be converted into the corresponding alcohol without inversion or racemization by known methods of ester cleavage (S. J. Salomon, E. G. Mata, O. A. Mascaretti, Tetrahedron 1993, 49, 3691-3748). Conversely, the resulting alcohol can be converted into the corresponding ester without inversion or racemization by known methods of esterification (Haslam, Tetrahedron 1980, 36, 2409).
The products resulting from or remaining in the process can be racemized and employed again in the racemate resolution according to known methods, e.g. by metal-catalyzed rearrangements (L. E. Overman, Angew. Chem. 1984, 96, 565-573 and literature already cited). This increases the yield to over 50%. For example, the compounds of the formula (I) where R=COR1 can be racemized directly and those of the formula (I) where R=H can be racemized, for example, after conversion into suitable derivatives, such as described in L. E. Overman, Angew. Chem. 1994, 96, 565-573. Metal catalysts which can be used are, for example, Hg(II), Pd(O) or Pd(II) compounds or salts.